Inflammation is the body's defense response which is induced by antigenic stimulation. An inflammatory response may worsen pathologically when inflammation takes place even after the removal of infectious antigenic substances or an inflammatory response is induced by an inappropriate stimulus such as an auto-antigen. Such an inflammatory response involves a variety of cytokines. In particular, as a cytokine which serves to control inflammation, a tumor necrosis factor (hereinafter, referred to as “TNF”) was identified.
TNF was originally discovered as a protein which eliminates tumor cells (Carswell et al., PNAS 72:3666-3670, 1975; Old et al., Science 230: 630-632, 1985; and Beutler et al., Nature 316:552-554, 1985). TNF is a class of cytokines produced by numerous cell types, including monocytes and macrophages, and is directly involved in inflammatory responses. At least two TNFs (TNF-α and TNF-β) have been previously described, and each is active as a trimeric molecule and is believed to initiate intracellular signaling by crosslinking receptors (Engelmann et al., J. Biol. Chem., 265:14497-14504). TNFs induce inflammatory responses in vivo to regulate cell-mediated immune responses and defense mechanisms and have important physiological effects on a number of different target cells (Selby et al., Lancep 1:483, 1988; Starnes et al., J. Clin. Invest 82:1321, 1988; Oliff et al., Cell 50; 555, 1987; Waage et al., Lancept 1:355, 1987; and Aggarwal et al., Nat. Rev. Immunol. 3:745-756, 2003). However, it was demonstrated that an excess of TNFs results in a pathological condition such as rheumatoid arthritis, degenerative arthritis, psoriasis or Crohn's disease, and suppression of TNFs exhibits therapeutic effects on the diseases (Feldmann et al., Nat. Med. 9:1245-1250, 2003; Kooloos et al., Drug Discov. Today 12:125-131, 2007; Rutgeerts et al., Gastroenterology 126:1593-1610, 2004; Rothe et al., Nature 364:798-802, 1993; Kafrouni et al., J. Leukocyte Biol. 74:564-571, 2003; Rahman et al., Plos Pathog. 2:e4, 2006; Chan et al., J. Biol. Chem. 278:51613-61621, 2003; and Wajant et al., Cell Death Differ. 10:45-65, 2003).
Tumor necrosis factor receptor (hereinafter, referred to as “TNFR”) is a cytokine receptor which binds to TNF.
Two types of TNFRs, known as p55-TNFRI and p75-TNFRII, have been currently discovered. Expression of TNFRI can be demonstrated in almost every mammalian cell while TNFRII expression is largely limited to cells of the immune system and endothelial cells.
The two TNF receptors exhibit 28% amino acid sequence similarity therebetween. Both receptors have an extracellular domain and have four cysteine-rich domains.
The cytoplasmic portion of TNFRI contains a “death domain” which initiates apoptotic signaling. TNFRII has no death domain and the function thereof has not been yet clearly defined. In addition, TNFRI and TNFRII exhibit a difference in terms of affinity for TNF-α which is a ligand. It is known that TNFRI exhibits an affinity 30 times or higher than that of TNFRII (Tartaglia et al., J. Biol. Chem. 268:18542-18548, 1993; and Bernard et al., Cytokine 7:759-770, 1995). Due to such affinity difference, a variety of attempts have been made for the development of pharmaceuticals regarding TNFRI.
TNFR adhering to the cell surface is cleaved by protease to produce soluble TNFR. The soluble TNFR neutralizes an excess of TNF to control the level of TNF. In cases such as autoimmune disease and chronic inflammation excessively high levels of TNF overwhelms the ability to self-regulate.
In order to artificially control TNF signaling, various strategies of blocking TNF have been attempted including inhibition of TNF synthesis, inhibition of TNF secretion or shedding, and inhibition of TNF signaling. Among TNF blocking methods, a method of blocking TNF signaling by preventing binding of TNFR to TNF has been applied for the development of pharmaceuticals. For example, etanercept, which is prepared by fusing a TNFRII extracellular region to the Fc region of an antibody, and antibodies capable of binding to TNF, adalimumab and infliximab have been used globally as a therapeutic agent for treating rheumatoid arthritis, psoriasis, ankylosing spondylitis, or the like.
Lenercept, which is a fusion protein of an antibody Fc to a TNFRI extracellular domain produced by applying the same technique as in the anti-rheumatoid arthritis drug etanercept, has completed a phase II clinical trial in Europe and USA (Furst et al., J. Rheumatol. 30:2123-2126, 2003; and Rau et al., J Rheumatol. 30:680-690, 2003 Arbraham et al., Crit. Care Med. 29:503-510, 2001). In addition, research has been carried out for a TNFRI dimer and a pegylated soluble TNFRI molecule (Carl et al., Ann. Rheum. Dis. 58:173-181, 1999; Solorzano et al., J. Appl. Physiol. 84:1119-1130, 1998; Carl et al., Advanced Drug Delivery Reviews 55:1315-1336, 2003; Honghui et al., J. Clin. Pharmacol. 45:490-497, 2005; and Yugal et al., The American Journal of Pathology 172:1411-1418, 2008).
Further, as an approach to reduce immunogenicity of TNFRI and increase the ability of TNFRI to bind with TNF, modification of amino acid sequences has been studied. In particular, a TNFRI mutant, against which the occurrence of an antibody has been decreased through partial substitution of the amino acid sequence of TNFRI, and a TNFRI mutant, which has an increased ability of TNFRI to bind with TNF, are known (U.S. Pat. No. 7,144,987).
Research has been actively made to find an active site responsible for binding of TNFR to TNF, and it is known that the fourth domain of TNFR is not essential for binding with TNF, and when deletion of the second and third domains results in loss of TNF binding activity (Corcoran et al., Eur. J. Biochem. 233:831-840, 1994; Chin-Hsuch et al., J. Biol. Chem. 270:2874-2878, 1995; and Scallon et al., Cytokine 7:759-770, 1995). Further, a certain region of the third domain for binding of TNFRI to TNF may be made deficient, and the amino acid sequence consisting of amino acid residues 59 to 143 of a human TNFRI polypeptide (SEQ ID NO: 1) is known to be a region showing a biological activity of TNFRI (U.S. Pat. No. 6,989,147).
Therefore, since binding of TNFRI to TNF is made in this region, other regions may include considerable added groups, eliminated groups or substituted groups.
Meanwhile, in order to enhance bioavailability, TFNRI is used in the form of a TNFRI polypeptide fragment rather than full-length TNFRI. For the purpose of producing an effective injection and oral formulation capable of minimizing protease cleavability and enhancing cellular permeability, TFNRI needs to be prepared as small in size as possible.
In addition, since protein therapeutics are cleared by general processes such as metabolism during in vivo circulation, glomerular filtration, and action of proteases in gastrointestinal tracts, tissues and blood, there is difficulty in delivery of a protein therapeutic to a target site while retaining an intrinsic activity of the protein in vivo. In particular, clearance of a drug by protease has significant effects on a half-life of a protein therapeutic upon administration thereof via oral administration, vascular injection, intramuscular injection, or the like.
A human tumor necrosis factor inhibitor, which is one of protein therapeutic drugs and controls in vivo TNF, has been developed in the form of an injection, but the administration of an injection has problems associated with pain and risk of infection. Therefore, another approach is required such as reduction of injection frequency or oral administration. Enhancement of stability of a human tumor necrosis factor inhibitor is essential for this purpose, but protease-induced degradation constitutes a great obstacle thereto.
Accordingly, an important goal of the development of an orally administrable protein therapeutic is to develop a protein therapeutic exhibiting protease resistance while having biological activity.